Protegrin-2, a potential inhibitor for targeting SARS-CoV-2 main protease Mpro

Background SARS-CoV-2 variants continue to spread throughout the world and cause waves of COVID-19 infections. It is important to find effective antiviral drugs to combat SARS-CoV-2 and its variants. The main protease (Mpro) of SARS-CoV-2 is a promising therapeutic target due to its crucial role in viral replication and its conservation in all the variants. Therefore, the aim of this work was to identify an effective inhibitor of Mpro. Methods We studied around 200 antimicrobial peptides using in silico methods including molecular docking and allergenicity and toxicity prediction. One selected antiviral peptide was studied experimentally using a Bioluminescence Resonance Energy Transfer (BRET)-based Mpro biosensor, which reports Mpro activity through a decrease in energy transfer. Results Molecular docking identified one natural antimicrobial peptide, Protegrin-2, with high binding affinity and stable interactions with Mpro allosteric residues. Furthermore, free energy calculations and molecular dynamics simulation illustrated a high affinity interaction between the two. We also determined the impact of the binding of Protegrin-2 to Mpro using a BRET-based assay, showing that it inhibits the proteolytic cleavage activity of Mpro. Conclusions Our in silico and experimental studies identified Protegrin-2 as a potent inhibitor of Mpro that could be pursued further towards drug development against COVID-19 infection.


Introduction
Since the beginning of the COVID-19 pandemic, several variants have appeared with enhanced virulence, increased transmission capabilities, or potential for immune escape [1,2]. The main protease (M pro , also known as 3CL protease), which is conserved among all the variants, is crucial for SARS-CoV-2 life cycle. Hence, the M pro is a potential target for the development of antiviral drugs [3]. Antimicrobial peptides (AMPs) have been extensively explored for their potential antiviral activities in various infections in recent years [4][5][6][7][8]. They are biologically active molecules produced by a wide variety of organisms, including humans, as an essential component of their innate immune response against invading pathogens. AMPs are short sequence peptides ranging from 10 to 100 amino acids, positively charged and amphiphilic. The Antimicrobial Peptide Database (APD) consists of 3425 AMPs, among which 200 are antiviral [9]. The mode of action of these peptides varies, including the blockage of interactions, direct inhibition of virus, and inhibition of absorption. Some AMPs also reduce the expression of viral genes [10]. Moreover, the positively charged residues on AMPs interact with negatively charged residues on the cell surface, such as heparan sulfate, which contains glycosaminoglycans that are associated with viral attachment. AMPs can also inhibit the spread of cell-to-cell infection and formation of syncytium [4]. These peptides also have great efficacy, with minimal toxicity in humans, and hence excellent specificity and effectiveness can be achieved at low dosages with little side effects [10]. Therefore, we explored the antiviral activities of naturally occurring antiviral peptides through their ability to inhibit M pro . In this study, we predict that Protegrin-2 can strongly bind to M pro and show it to inhibit the activity of M pro in vitro.

Preparation of the protein and peptides
The 3D structure of M pro was downloaded from the RCSB Protein Data Bank (PDB ID: 7LKD) [11]. A total of 200 reported antiviral peptides of different species were selected from the antimicrobial peptide database (APD) [9]. The 3D structures of 62 peptides were retrieved from PDB, whereas the 3D structures of 138 peptides were designed using AlphaFold [12]. Pymol 1.7.4.5 was used for geometry optimization and minimization of all the selected peptides and the protein [13].

Protein-peptide docking studies
HDock server [14] was used to find the binding interactions of M pro with the selected peptides. The docking prediction jobs for all the 200 peptides were submitted to the server by using the template free docking option. The best pose complexes with low binding energies were selected for the interaction analysis. For the interaction analysis and visualization, PDBsum [15] and Pymol version 1.7.4.5 [13] were used.

Molecular dynamics (MD) simulation
Molecular dynamics simulations of apo-and holo-M pro were carried out using Gromacs version 5.1.4 [21]. Ligand topology files were created using automated topology builder (ATB). The complete protein complexes were solvated in three-dimensional box with a size of 534.76 nm 3 along with simple point charge (SPC) water. Three Na + ions were added in the solution. Steric overlap was eliminated by using sharpest descent algorithm, and each system was subjected to 5000 energy minimization steps with a cut-off > 1000 kJmol − 1 . Following that, all the systems underwent a two-stage equilibration phase called NVT and NPT. The system's temperature was stabilized for 500 picoseconds (ps) using the NVT equilibration, and the pressure was stabilized for 500 ps using NPT. The V-rescale temperature-coupling [22] method was employed for the NVT system, whereas the Nose-Hoover pressure coupling [23] was used for NPT with a temperature of 303.15 K at 1 ps. The Particle Mesh Ewald method was used for the calculation of electrostatic forces for both the NVT and NPT systems [24]. Finally, all the systems were subjected to a full 100 nanoseconds (ns) simulation. Analysis of the MD trajectory files was carried out using the GROMACS tools. The root mean square deviation (RMSD) was determined using gmx rmsd whereas the calculation for root mean square fluctuation (RMSF) was done using gmx rmsf. Moreover, the radius of gyration (Rog) and the number of hydrogen bonds were determined using gmx gyrate and gmx hbond.

In vitro, BRET-based M pro proteolytic cleavage inhibitor assay
Recombinantly purified SARS-CoV-2 M pro protease (500 nM) was incubated with various concentrations (ranging from 10 − 6 to 10 − 12 M) of Recombinant Pig Protegrin-2 (NPG2-CSB-EP341296PI, 20 microgram) in a buffer containing Tris-buffered saline (TBS), 1 M sodium citrate, 1 mM EDTA and 2 mM DTT for 2 h at 37 • C. GC376 (GC376 Sodium; AOBIOUS -AOB36447; stock solution prepared in 50 % DMSO at a concentration of 10 mM) served as control. The cell lysate containing mNG-M pro -Nter-auto-NLuc sensor was added and further incubated for 2 h at 37 • C. BRET measurements were performed at 37 • C by the addition of furimazine (Promega, Wisconsin, USA) at a dilution of 1:200. The bioluminescence (467 nm) and fluorescence (533 nm) readings were recorded using Tecan SPARK multimode microplate reader and used to calculate the BRET ratios (533 nm/467 nm).

Molecular docking of M pro with antimicrobial peptides
Docking of the M pro protein with the 200 antiviral peptides present in the AMP database revealed the differences in binding affinities (Supplementary Table 1). Out of these peptides, Protegrin-2 (PDB ID: 2MUH) [28] exhibited the highest binding affinity with M pro (HDock scores: − 216.84 kcal/mol) (Fig. 1). Protegrin-2 showed the highest binding potential at the allosteric site, forming eight hydrogen bond interactions with the side residues (D176, Q110, F294, Y154, N151, I152 and D153) of M pro . Hence, we took forward Protegrin-2 for further detailed analysis as described below.

Molecular dynamics simulation of M pro with protegrin-2
To assess the stability of binding of Protegrin-2 with M pro , we performed MD simulations of the docked complex ( Fig. 2A and Fig. 3). Trajectory analysis of the root-mean-square deviation (RMSD) values of the backbone indicated that the M pro -Protegrin-2 complex reached equilibrium during the 100 ns simulation and the low RMSD values showed a stable complex formation (Fig. 2B). The RMSD of holo-M pro with Protegrin-2 between the first frame at 0 ns (F0) and the last frame at 100 ns (F100) reached 0.3 nm, and it was only 0.4 nm when compared with the frame at 50 ns (F50) to F100 (Fig. 2B). Moreover, the root-mean-square fluctuation (RMSF) revealed a region of high dynamic fluctuation in apo-M pro between residues aa 50 -150 (Fig. 2C). The radius of gyration (Rog) of M pro -Protegrin-2 first decreased between 0 and 20 ns and after the 20 ns increased to reach 2.15 nm followed by a sharp decrease to reach 2.1 nm at 80 ns and then stable up to 100 ns (Fig. 2D). The complex showed a high value of solvent-accessible surface area (SASA) between 0 ns and 50 ns compared to the apo-M pro (Fig. 2E). The number of hydrogen bonds in the M pro -protegrin-2 complex increased between 0 and 100 ns as compared to apo-M pro (Fig. 2F). Importantly, we found a sustained H-bond between Protegrin-2 and the Q110 residue in M pro with an occupancy of 70.68 %, whereas the residue N151 of M pro formed hydrogen bonds with Protegrin-2 with occupancies of 73.26 %.

Inhibition of M pro activity with protegrin-2
Based on our computational results discussed above, we proceeded to determine the impact of Protegrin-2 on the catalytic activity of M pro . The inhibitory potential of Protegrin-2 on the catalytic activity of the M pro was determined by in vitro assays using an M pro sensor that we recently developed [29] (Fig. 4A). This sensor is based on bioluminescence resonance energy transfer (BRET), wherein the efficiency of energy transfer between a donor and acceptor largely depends on the distance between the two proteins [29][30][31][32][33][34]. The sensor consists of a green fluorescent protein, mNeonGreen, that serves as the resonance energy acceptor and a bioluminescent protein, NanoLuc, that serves as the energy donor sandwiching the cognate M pro N-terminal autocleavage site. In the absence of M pro -mediated cleavage of the sensor, a high resonance energy transfer is observed while M pro -mediated cleavage of the peptide results in a substantial decrease in resonance energy transfer, as determined from the ratio of emission at 533 nm (mNeonGreen emission) and 467 nm (NLuc emission). For this, we expressed the M pro sensor in HEK 293 T cells and prepared cell lysates while SARS-CoV-2 M pro was expressed and purified from bacteria. Incubation of the sensor with the recombinantly purified M pro resulted in a decrease in BRET, while inclusion of the known M pro inhibitor, GC376 (100 μM), resulted in the abrogation of the decrease in BRET (Fig. 4B). Further, incubation with a range of GC376 concentrations resulted in a dose-dependent decrease in M pro proteolytic activity as indicated by the increase in BRET (Fig. 4C), with an IC 50 value (0.9 ± 0.1 μM) similar to that reported previously [47-51]. We then performed dose-response experiments with Protegrin-2 and found the peptide to inhibit M pro with high affinity, with an IC 50 values of 0.83 ± 0.07 nM as compared to GC376 (Fig. 4D,E).

Discussion
We have identified Protegrin-2 as a potential inhibitor of the SARS-CoV-2 main protease M pro through large-scale in silico and focused in vitro studies. COVID-19 reached pandemic proportions due to the emergence of SARS-CoV-2 variants such as B.1.1.7, B.1.1.529, BA.2 with increased transmissibility and the ability to evade immune response against the spike (S) glycoprotein, which helps in the virus entry to the host cell. The main protease M pro , which is largely conserved among several SARS-CoV-2 variants, provides an alternate target for inhibition of the virus [35]. Naturally occurring antimicrobial peptides derived from various sources have been found to be effective against viruses and a few against SARS-CoV-2 as well, including LL-37 (cathelicidin) [36], protegrin-1 [36], P9R [37], beta-defensin 1 [36], human intestine defensin-5 [38,39].
High selectivity, safety, tolerability, and efficacy of peptides have attracted the interest of researchers in developing peptide-based treatments [40,41]. The identification of anti-SARS-CoV-2 activity in numerous naturally and synthetically created peptides targeting viral attachment and replication reinforces the need for peptide-based prophylactics and treatment against SARS-CoV-2. However, there are also potential downsides to peptide-based treatments, such as chemical and physical instability, sensitivity to proteolytic hydrolysis, a tendency for aggregation, and limited bioavailability and membrane permeability [42,43]. Several ways of overcoming these constraints have been proposed, such as the modification of both the side chains and the amide bond which can result in proteolytically resistant peptidomimetics. Moreover, the incorporation of D-amino acids into the peptide causes cyclization, which confers resistance to proteolytic breakdown and enhances absorption following oral administration [40,42]. Such methodologies can aid in the development of safe, efficient, and effective peptide-based vaccines and therapies.
Protegrin-2 (NPG-2) is a cysteine rich potent antimicrobial peptide originally isolated from pigs [44]. Two cysteine-cysteine disulfide bridges serve to stabilize the anti-parallel hairpin conformation of Protegrin-2, which greatly enhances its antimicrobial activity [45]. This cyclic peptide has low toxicity, and the conformational rigidity and increased surface area of cyclic structures contribute to their high selectivity and increased affinity [46]. Furthermore, our BRET-based assay provided a clear indication of the inhibitory activity of Protegrin-2 on SARS-CoV-2 M pro , suggesting its potential use against COVID-19. However, further in vivo studies are required to validate the in vitro results presented here and determine the utility of the peptide as a therapeutic agent against COVID-19.

Conclusion
In summary, this study identified a potential M pro inhibitor by combining both in silico and in vitro approaches. This work experimentally validates Protegrin-2 as a potential antiviral drug to treat COVID-19 infection. This research advances the development of peptidesbased effective antiviral medicines for COVID-19 infection.

Ethics approval statement
Ethical approvals were not required for this study.